Rearing
All mosquitoes used in these experiments were derived from a laboratory-reared colony of An. stephensi is initially established (six generations) in University College Agriculture, University of Sargodha. Uninfected mosquitoes were maintained in the laboratory; in gauze-covered boxes (30 cm wide × 30 cm high × 50 cm deep) under control condition 27 ± 2 °C temperature and 75–80% relative humidity. Auto ON/OFF switches with the timer were used to break the scotophase (dark) period in the control conditions of the laboratory with the light: dark cycle set to 12:12 h48. A 10% fructose solution supplemented with 0.05% para-minobenzoic acid (PABA) was provided to mosquitoes. The adult mosquitoes were reared on blood provided via an in situ electronically derived artificial membrane feeder, set at 37 ± 1 °C, and offered twice a week, and offered twice a week given their need for another blood meal approximately 5–6 h after the first49. Multiple blood feeding is vital for Anopheline species as it has been demonstrated as influencing reproductive behavior50. Oviposition cups were provided two days following the second blood meal. The larvae were reared under laboratory conditions described above and provided a certified Laboratory Rodent Diet (LRD) Lab Diet 500151.
Fecundity and fertility
To determine differences in fecundity (number of eggs) and fertility (percentage of fertile eggs) in An. stephensi mosquitoes, cages of mosquitoes were provided ABO blood groups and control (distilled water) via artificial membrane feeders (as described above for mosquito rearing). The blood was obtained from the blood bank of DHQ (one batch of each blood group was used for each replication), Chakwal Punjab, Pakistan. A total of 10 replicate cages were used for each blood type and control, so 10 batches of each blood group (ABO) (just to reduce the potential individual to individual variation) were obtained from the DHQ. After each replication along with the new batch of each blood group, a new strain of mosquitoes was used just to reduce the learning behavior of mosquitoes. Feeding success was determined by calculating the percentage of fed mosquitoes, and also, the numbers of fully engorged female mosquitoes were recorded.
To determine fecundity, females from each blood group were removed, killed, and dissected under a microscope, and the numbers of eggs per female were counted 60 h post-blood-feeding. Additionally, to determine oviposition and larval development, 40 fully fed female mosquitoes were caged in one of three replicate glass cages with gauze (25 cm wide × 25 cm high × 25 cm deep) and provided wet filter papers were placed for egg-laying. The total number of eggs was counted after every 12 h from 48 h until 96 h post-blood-feeding under a light microscope. The total numbers of eggs/40 females/box for each human blood group for 10 replicates were calculated.
For fertility estimation, an additional 40 gravid An. stephensi from each treatment and replication, including the control group, the females were gently transferred to the cages with triangular Whatman filter paper No. 1 using the mouth aspirator. The egg laid in each experimental and control group was reared in plastic trays filled with distilled water. The numbers of hatched larvae were recorded for a fertility test. While the eggs that could not be hatched into larvae up to day 7 were considered as infertile. The number of fertile and infertile eggs was recorded from all experimental and control cages.
The collected eggs from each experimental box were placed into the plastic trays (24 cm wide × 12 cm high × 7 cm deep) with water, and the development of mosquitoes was observed until adult mosquitoes had emerged from all pupae for each blood group. The water in these plastic trays was maintained at a constant level throughout immature mosquito rearing. The larvae were fed a certified Laboratory Rodent Diet (LRD) Lab Diet 500151. The rearing was done according to the standard mass rearing of Anopheles techniques52. Pupae were counted and removed from the tray and placed in cages according to each human blood group type fed to allow emergence, and the percent of male and female mosquitoes was recorded. Adult mosquitoes were maintained on a 10% fructose solution supplemented with 0.05% para-minobenzoic acid (PABA) but were not provided with a blood meal. The mortality of adult mosquitoes was recorded daily until total mortality reached 100%.
Digestibility tests
The precipitin and benzidine tests were used to test the effect of human blood groups ABO (on the rate of digestion in mosquitoes). The experiment was conducted in controlled laboratory conditions where the temperature, humidity, and day and night periods were maintained as described above. Mosquitoes (10 mosquitoes were used for each blood group and the same experiment was repeated 10 times) that had not been fed previously on either a sugar solution or blood were used in experiments. Mosquitoes were provided one of four different human blood group types, as previously described. After feeding the female were kept in the same boxes without any further food and water, and boxes were placed in an incubator where the temperature and the relative humidity was at a constant level (28 ± 2 °C and 80 ± 5%). The engorged female adult mosquitoes were killed at 8 h intervals, rubbed over the filter paper53, and the filter papers were placed inside the refrigerator until the test could be conducted. Approximately 48 female mosquitoes were used in each boxed marked for each blood group. The rate of blood digestion in the engorged blood was classified according to the Sella scale, following Detinova et al.54.
To perform the precipitin test, the physiological saline and the filter paper smears were extracted in a small capillary tube. The specific antiserum was also extracted in the same capillary tube at the end; the change in color, clumping, and cloudiness of the solution indicates the presence of human blood in the tissue smears55. The collected material was heated in a steam oven for 10–12 min to apply the benzidine test at 108–110 °C. The test was used to check the traces of iron porphyrins in the abdomen of mosquitoes.
Effect of blood groups on oogenesis
To test the blood-specific effects on the development of the ovaries of An. stephensi, the ovaries of fully fed female mosquitoes were collected separately from the box of each blood group 36 h post engorgement. For scanning electron microscopy (SEM), the whole female mosquitoes were selected from each box of every blood group separately (10 females for each blood group ABO). In preparing specimens for the scanning electron microscope, the process is divided into two fixations, the primary and the secondary fixation. For the primary fixing process, the 2.5% glutaraldehyde in 0.1 M cacodylate buffer was used for the period of 2 h, followed by the three consecutive washing with the same buffer for the 30 min. While for the secondary fixation process, 1% osmium tetroxide was used for the 2 h. Then the samples were rinsed for the final time with the 0.1 M cacodylate buffer three times for 30 min.
The ovaries were dehydrated by using the graded series of acetone (50%, 70%, 80%, 90%, and 100%). The dehydrated ovaries were then transferred to the critical point drying apparatus. The recommended quantity of acetone solution was also poured into the drying chamber to avoid over-drying. Liquid nitrogen was also added into the drying critical point drying chamber. The CO2 and acetone were allowed to be mixed freely; the same process was repeated eight times to confirm the drying of the specimen. The dried mosquito specimens were mounted over the stubs, and the specimens tubes were coated with a thin layer of silver. Gold-spotted SCD005 was used, and then the samples were photographed with SEM.
Electroantennography (EAG)
To measure the response of mosquitoes to each human blood group type, EAG recordings from one antenna of An. stephensi female mosquito was made. Unfed female mosquitoes were anesthetized by the use of CO2 and were permanently fixed with the reference electrode by the use of spectra 360 electrode gel. It was made sure that the mosquito was completely immobile except for the antennae. The tips of the antennae were pressed into the small drop of electrode gel on the recording electrode. Both of the electrodes are silver wires coated with silver chloride with a diameter of 0.2 mm. The experimental preparations were done in continuous airflow (600 mL/min, 1.5 m/s) by the Teflon tube of 0.7–0.8 cm diameter, containing about 100 mL/min dry air and the 600 mL/min moist air passed through the charcoal filter. At this stage, little modification was done in the structure of the electroantennogram, and an artificial blood feeder of mosquitoes with the membrane was attached to the system.
The blood feeder was packed in a glass jar through which continuous airflow was passed, and this air flow ends as stimuli near the mounted mosquito. The diameter of the glass tube was 0.5 mm, and the flow of air was controlled by using an ON/OFF switch; three bursts of 0.5 s of air from the blood jar were provided as a stimulus to host-seeking mosquitoes. All the blood groups were tested systematically together with the control group. The amplifier amplified the generated signals while the well-known software decoded the recordings (EAG 2000, Syntech, Hilversum, and the Netherlands). All of the test blood groups were also dissolved separately in tetryl-butyl-methyl ether (MTBE), and about 30 uL of this test solution was applied onto a piece of filter paper (1.5–2 cm). About 20 min was given to the TMBE solution to evaporate from the filter paper leaving behind the blood; then, this piece of filter paper was placed into the Pasteur pipette. In the case of the control treatment, distilled water was used, and the same treatment was applied with the distilled water for the test compounds. The stimulus controller C5-01/b, Syntech, was used to inject the odor cues originating from the treated filter paper in the Pasteur pipette into the humidified and filtered air stream directed towards the antennae of immobilized mosquitoes. Olfactory stimuli were tested randomly against different mosquito specimens with a total of five specimens exposed to each of the human blood type groups and control.
To minimize the chances of error and to test the electrophysiological activity of the stuck female mosquito, lactic acid, 1-octen-3-01, and isovaleric acid were used as known stimulants41,56. After that, each blood group was replicated three times to record the activity of the olfactory neurons of antennae. All the treatments of blood groups were tested randomly, and a regular interruption of control stimulus (0.1% lactic acid) was done. The regular interruption of the control stimulus was used to control the activity of the antennae. All the stimulants were expressed as a mean percent response to the control treatment. The response of different female mosquitoes to human blood groups was indicated as a mean percent response. The results were analyzed by the use of the Student’s t-test.
Wind tunnel bioassays
Wind tunnel bioassays were used to determine the response of An. stephensi to four human blood groups (A, B, AB, and O) and control stimuli (distilled water). Wind tunnel bioassays have been used to evaluate the response of Ae. aegypti, Cx. quinquefasciatus and Cx. nigripalpus towards the blood volatiles44. A dual choice wind tunnel was converted into a “five-choice” tunnel with all five glass tubes having glass jars at their end with openings to accommodate an artificial blood feeder. A continuous flow of warm water ensured the blood remained in liquid and produced its specific smell.
A batch of approximately 100 female mosquitoes was released at the downwind end of the tunnel in the air stream coming from the five upwind end chambers. After 30 min, the numbers of mosquitoes in each of the five glass jars were counted. The mosquitoes were then sent back towards the downwind end of the wind tunnel, and the positions of odor cues were changed, including the control. Before the second time release, the fresh air was passed through the tunnel. Again the mosquitoes were released from the releasing box, and the response of the mosquitoes towards the new cues and the number of mosquitoes in each chamber at the upwind end was counted after 30 min; the same process was repeated, and for the third time with randomization. The same process was repeated 10 times with each blood group and with a new batch of mosquitoes each time.
To test the response of female mosquitoes towards human-emitted olfactory cues, an olfactometer was used in previous studies41,43. The olfactometer test was conducted in the control room; the temperature was 27 ± 2 °C with 70–80% relative humidity. The optimum activity of the An. stephensi was observed late at night, so the experiment was conducted at 2–6 AM57.
Steel balls rubbed in the hands of persons (ten volunteers per blood group) of having ABO blood groups along with the few drops of blood group-specific sweat were placed in the glass jars at the upwind end of the olfactometer. Approximately 100 female mosquitoes were released at the downwind end of the olfactometer from the releasing cage. After 30 min, the total number of mosquitoes in each box at the upwind end was counted, including the control. After that, the mosquitoes were returned to the releasing cage; then, the positions of steel balls at the upwind end of the glass jar of the olfactometer were changed randomly to decrease the biases from the data. To remove the smell of a sweat from the olfactory tube after cleaning, the fresh air was passed for about 10 min continuously. Mosquitoes were then again allowed to enter into the olfactometer, and after 30 min, the total number of mosquitoes was counted. The same process was repeated for the third time. The same experiment was repeated 10 times with different persons and mosquitoes to decrease the chances of error. A new batch of mosquitoes was selected for each replication.
Statistical analysis
The mean number of eggs of An. stephensi were evaluated with the help of a linear model (ANOVA) and Tukey’s test on Minitab® software (12.2, version, Minitab). Before performing the ANOVA, with the help of angular transformation (arcsine √x), egg viability was also transformed58 for fertility. Data obtained were analyzed using R 3.2.2 software. The Shapiro–Wilk normality test was carried out, which showed that the data were not normally distributed. Hence, the Kruskal–Wallis Chi-square test was used to compare the averages of the responses of An. stephensi in relation to blood-group treatments.
Ethics statement
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
Source: Ecology - nature.com
